Identification of ERK5 as a miR-143 target.

<p><i>A</i>, Schematic representation of the predicted target site of miR-143 in the 3′ UTR of ERK5 mRNA. Seed-matching sequence is indicated. H.s ERK5 3′UTR is the Homo Sapiens 3′UTRsequence. <i>B</i>, Western blot analysis of endogenous ERK5 expression in prostate normal and cancer cell lines. The relative expressions, normalized to GAPDH, were measured by Image J software as a fold of the indicated situation versus scrambled miR. Data are means ± SEM for three independent experiments. C, Representative immunohistochemical staining of ERK5, and in situ hybridization of miR-143 in consecutive sections of high-density tissue array. <i>D</i>, Western blot analysis of endogenous ERK5 expression in C4-2 and LNCaP cells 48 h after transient transfection of the indicated miR-143. Relative expression, quantified by Image J software is normalized to GAPDH, and measured by fold of the indicated situation versus scrambled miR. Data are means ± SEM for three independent experiments. <i>E</i>, Measure of the luciferase activity in COS cells transfected with a reporter luciferase gene pGL3 fused to the ERK-5 3′ UTR mutated or not with increasing concentrations or miR-143 (0; 25; 50; 100 nM). Values are normalized to beta-galactosidase activity and expressed in fold versus absence of miR-143. Black bars, ERK5 3′UTR, white bars, mutant 3′ UTR; data are means ±SEM for four experiments conducted in triplicate. <i>F</i>, Western blot analysis of endogenous c-Jun expression in LNCaP cells 48 h after transient transfection of the indicated miR-143. Data are representative for three independent experiments. <i>G</i>, QPCR of ERK5 in C4-2 cells (white bars) and LNCaP cells (black bars), normalized to 18 s gene 48 h after transfection with pSuper-shNeo (control) or pSuper-shERK5. Data are means ± SEM for three independent experiments. <i>H</i>, Quantification of BrdU incorporation in C4-2 cells (white bars) and LNCaP cells (black bars) 48 after transfection with pSuper-shNeo (control) or pSuper-shERK5. Data are representative for three independent experiments.